Transcriptional regulation and expression of guanylyl cyclase/natriuretic peptide receptor‐A gene by δEF1:Role of TGF‐β1 signaling

Abstract

The binding of atrial and brain natriuretic peptides (ANP and BNP) to guanylyl cyclase‐A/natriuretic peptide receptor‐A (GC‐A/NPR‐A) produces second messenger cGMP, which plays a critical role in various intracellular signaling pathways including; hypertension, cardiovascular events, and cancer. Transcription factor δEF1, an E‐2 box repressor has been predicted to have binding sites in the 5′ flanking region of the Npr1(coding for GC‐A/NPRA) gene promoter. The objective of this study was to determine the repressive effect of δEF1 on transcriptional regulation of murine Npr1 gene promoter in mouse mesangial cells (MMCs) and rat thoracic aortic vascular smooth muscle cells (RTASMCs). MMCs were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and ITS (insulin, transferrin, and selenium). RTASMCs were cultured in DMEM containing 10% FBS. Co‐transfection of δEF1 expression plasmid with the Npr1 gene promoter‐luciferase constructs showed 70–75% reduction in the luciferase activity in MMCs and RTASMCs. Also, Npr1 gene promoter‐luciferase constructs embodying δEF1 sites showed up to 90 % reduction in luciferase activity after treatment with TGF‐β1 in a time‐ and dose‐ dependent manner. Deletion of δEF1 sites eliminated the repression of luciferase activity induced by TGF‐β1. Electrophoretic mobility shift assay as well as supershift assay confirmed the binding of δEF1 to the specific sites of Npr1 gene promoter. The findings of this study suggest that δEF1 exerts a repressive effect on Npr1 gene transcription and expression, which plays a critical role in hypertension and cardiovascular regulation. This work is supported by National Institutes of Health Grants (HL57531 and HL 62147).